Antibiotic 107891, its factors A1 and A2, pharmaceutically acceptable salts and compositions, and use thereof

ABSTRACT

The invention relates to an antibiotic substance of microbial origin, arbitrarily denominated antibiotic 107891 which is produced by fermentation of  Microbispora  sp. ATCC PTA-5024, the pharmaceutically acceptable salts and compositions thereof, and their use as an antibacterial agent having inhibitory activity versus susceptible microbes. Antibiotic 107891, which is a complex comprising two Factors, denominated Factors A1 and A2, has a peptide structure containing lanthionine and methyllanthionine as constituents which are typical characteristics of the antibiotics of the lantibiotics group. Antibiotic 107891 and its Factors A1 and A2 show a good antibacterial activity against Gram-positive bacteria including methicillin resistant and vancomycin resistant strains, and is active also against some Gram-negative bacteria such as  M. catharralis, Neisseria  species and  H. influenzae  and Mycobacteria.

This application is a continuation-in-part of U.S. application Ser. No.11/035,296, filed Jan. 12, 2005, which is a continuation-in-part of U.S.application Ser. No. 10/521,336, filed on Jan. 11, 2005, which is a §371 national filing of PCT/EP2004/007658, filed on Jul. 12, 2004, whichclaims priority to EP Application No. 03016306.7, filed Jul. 18, 2003,all of which are hereby expressly incorporated by reference in theirentirety.

The present invention concerns an antibiotic substance of microbialorigin, arbitrarily denominated antibiotic 107891, which is a complexcomprising Factors A1 and A2, the pharmaceutical acceptable saltsthereof, pharmaceutical compositions thereof and their use as anantibacterial agent.

Another object of the present invention is a process for preparingantibiotic 107891 which includes culturing Microbispora sp. 107891(hereinafter identified as Microbispora sp. ATCC PTA-5024) or a variantor mutant thereof maintaining the ability to produce said antibiotic,recovering the antibiotic of the invention from the mycelium and/or fromthe fermentation broth, isolating the pure substance by chromatographicmeans and separating Factors A1 and A2.

Antibiotic 107891 is a novel antimicrobial agent with a peptidestructure containing lanthionine and methyllanthionine as constituents.These are the typical characteristics of lantibiotics and, inparticular, of the subgroup acting primarily on cell wall biosynthesis.

Lantibiotics are peptides, which contain the thioether amino acidlanthionine as well as several other modified amino acids (H. G. Sahland G. Bierbaum, (1998) “Lantibiotics: biosynthesis and biologicalactivities of uniquely modified peptides from gram-positive bacteria”,Ann. Rev. Microbiol. 52:41-79). The majority of known lantibiotics haveantibacterial activity, although some have been reported as active ondifferent pharmacological targets. The antibacterial lantibiotics can bebroadly divided into two groups on the basis of their structures: type-Alantibiotics are typically elongated, amphiphilic peptides, while type-Blantibiotics are compact and globular (O. McAuliffe, R. P. Ross and C.Hill, (2001): “Lantibiotics: structure, biosynthesis and mode ofaction”, FEMS Microb. Rev. 25: 285-308). Nisin is the typicalrepresentative of type A lantibiotic, whereas actagardine (gardimycin)and mersacidin belong to the type B lantibiotic subclass. Bothnisin-type and mersacidin-type lantibiotics interact with themembrane-bound peptidoglycan precursors lipid II, although the twoclasses differ in the effects they produce in the bacterialproliferation process. Nisin-type lantibiotics primarily kill bacteriaby permeabilization of the cytoplasmic membrane (H. Brotz, M. Josten, I.Wiedemann, U. Schneider, F. Gotz, G. Bierbaum and H. G. Sahl, (1998):“Role of lipid-bound peptidoglycan precursors in the formation of poresby nisin, epidermin and other lantibiotics”, Mol. Microbiol. 30:317-27),whereas mersacidin-type lantibiotics primary kill the bacterial cell byinhibiting the cell wall biosynthesis (H. Brotz, G. Bierbaum, K.Leopold, P. E. Reynolds and H. G. Sahl, (1998): “The lantibioticmersacidin inhibits peptidoglycan synthesis by targeting lipid II”,Antimicrob Agents Chemother. 42:154-60).

Two antibiotics produced by Microbispora corallina strain NRRLL 30420,identified as antibiotic MF-BA-1768α₁ and MF-BA-1768β₁, respectively,are described in U.S. Pat. No. 6,551,591 B1. The physico-chemical datareported in the above-identified patent (e.g. mass spectroscopy data,molecular weight, content of aminoacids) and comparison of the retentiontimes in LC-MS experimental analyses clearly show that the antibiotic107891 complex as well as its components Factor A1 and Factor A2 arechemical entities distinct from antibiotics MF-BA 1768α₁ andMF-BA-1768β₁.

EP 0592835A2 describes antitumor antibiotics BU-4803TA₁, A₂, B, C₁, C₂and D. Antibiotics BU-4803TA₁ A₂, and B are recovered from thefermentation broth of Microbispora ATCC 55327 (AA 9966) whileantibiotics BU4803TC₁, C₂ and D are products of transformation ofantibiotic BU 4803TA₁, A₂ and B, respectively, when these products arestored in dimethyl sulfoxide. The physico-chemical data reported in EP0592 835 A for the above antibiotics (e.g. aspect, U.V. absorbtion,molecular weight, antitumor activity, clearly show that they arechemical substances distinct from antibiotic 107891 complex and itsFactors A1 and A2.

Strain and Fermentation

Microbispora sp. 107891 was isolated in the environment and deposited onFeb. 27, 2003 with the American Type Culture Collection (ATCC), 10801University Blvd, Manassas Va., 20110-2209 U.S.A., under the provision ofthe Budapest Treaty. The strain was accorded accession number PTA-5024.

The production of antibiotic 107891 is achieved by cultivating aMicrobispora sp. strain capable of producing it, i.e. Microbispora sp.ATCC PTA-5024 or a variant or mutant thereof maintaining the ability toproduce said antibiotic; isolating the resulting antibiotic from thewhole culture broth and/or from the separated mycelium and/or from thefiltered fermentation broth; and purifying the isolated antibiotic bychromatographic means. In any case, it is preferred to produceantibiotic 107891 under aerobic conditions in an aqueous nutrient mediumcontaining easy assimilable sources of carbon, nitrogen, and inorganicsalts. Many of the nutrient media usually employed in the fermentationfield can be used, however certain media are preferred.

Preferred carbon sources are sucrose, fructose, glucose, xylose, and thelike. Preferred nitrogen sources are soybean meal, peptone, meatextract, yeast extract, tryptone, aminoacids, hydrolized casein and thelike. Among the inorganic salts which can be incorporated in the culturemedia, there are the customary soluble salts capable of yielding sodium,potassium, iron, zinc, cobalt, magnesium, calcium, ammonium, chloride,carbonate, sulphate, phosphate, nitrate, and the like ions.

Preferably, the strain producing antibiotic 107891 is pre-cultured in afermentation tube or in a shake flask, then the culture is used toinoculate jar fermentors for the production of substantial quantities ofsubstances. The medium used for the pre-culture can be the same as thatemployed for larger fermentations, but other media can also be employed.The strain producing antibiotic 107891 can be grown at temperaturebetween 17° C. and 37° C., optimal temperatures being around 28-30° C.

During the fermentation, antibiotic 107891 production can be monitoredby bioassay on susceptible microorganisms and/or by HPLC analyses.Maximum production of antibiotic 107891 generally occurs after circa 90hours and before the 200 hours of fermentation.

Antibiotic 107891 is produced by cultivating Microbispora sp. ATCCPTA-5024 or a variant or mutant thereof capable of producing antibiotic107891, and it is found in the culture broths and/or in the mycelium.

In this description and claims the term “antibiotic 107891”, unlessotherwise specified, identifies the antibiotic 107891 complex comprisingFactors A1 and A2.

Morphological Characteristics of Microbispora sp. ATCC PTA-5024

Microbispora sp. ATCC PTA-5024 grows well on various standard solidmedia. Microscopic dimensions were measured using the culture grown onhumic acid-Trace Salts Agar (composition in g/l: humic acid 0.5,FeSO₄*7H₂O 0.001, MnCl₂*4H₂O 0.001, ZnSO₄*7H₂O 0.001, NiSO₄*6H₂O 0.001,MOPS 2, agar 20) added with 1 ml/l of vitamins solution (thiaminehydrochloride 25 mg/l, calcium pantotenate 250 mg/l, nicotinic acid 250mg/l, biotin 0.5 mg/l, riboflavin 1.25 g/l, cyanocobalamin 6.25 mg/l,paraminobenzoic acid 25 mg/l, folic acid 500 mg/l, pyridoxalhydrochloride 500 mg/l).

In liquid culture (V6 medium, composition in g/l: dextrose 22, meatextract 5, yeast extract 5, casein 3, NaCl 1.5) no fragmentation of themycelium is observed after 6 days of growth at 28° C. Microscopicexamination on Humic acid-Trace Salts Agar (after 21 days of incubationat 28° C.) reveals a branched, un-fragmented substrate mycelium and amonopodially branched aerial mycelium; many long, straight and poorlybranched aerial hyphae are also visible. Characteristic longitudinalpairs of spores are borne by short sporophores laterally arising frombranches or directly from the main aerial hyphae. Spores are globose andnon-motile. Sporangium-like bodies or other particular structures arenot observed.

Cultural Characteristics of Microbispora sp. ATCC PTA-5024

Microbispora sp. ATCC PTA-5024 was grown for six days in AF/MS liquidmedium (see Example 1) at 28° C. and 200 rpm, then transferred (5%inoculum) to a new AF/MS liquid medium and grown for further 6 days andfinally inoculated (7% inoculum) into 100 ml of V6 liquid medium (seeExample 1). After 6 days of growth at 28° C. and 200 rpm, the myceliumwas harvested by centrifugation and washed three times by sterile salinesolution, then diluted to provide a suitable inoculum. Aliquots of thesuspension were streaked in a cross-hatched manner onto various mediarecommended by Shirling and Gottlieb (E. B. Shirling and D. Gottlieb,(1966): “Method for Characterization of Streptomyces species”, Int. J.Syst. Bacteriol. 16: 313-340), and media recommended by S. A. Waksman(1961): “The Actinomycetes”, The Williams and Wilkins Co., Baltimore.Vol. 2: 328-334).

The ability to use a variety of carbohydrates as a carbon and energysource was determined using medium ISP4 without starch, added with 1ml/l of the vitamin solution described above as basal medium; eachcarbon source was added at the final concentration of 1% (w/v).

NaCl tolerance, pH range of growth as well as ability to grow atdifferent temperatures was determined onto ISP2 medium. All media wereincubated at 28° C. for three weeks; descriptions are referred to 21days unless specified. Colour was assessed in natural daylight, usingthe Colour Atlas of Maerz and Paul (A. Maerz and M. R. Paul, 1950—ADictionary of Colour, 2nd edition. McGraw-Hill Book Co. Inc., New York).Ability to reduce nitrates to nitrites was evaluated in sloppy Nitratemedium according to the procedure described by Williams et al. (S. T.Williams, M. Goodfellow, G. Alderson, E. M. H. Wellington, P. H. A.Sneath & M. J. Sackin, 1983—Numerical classification of Streptomyces andrelated genera—J. Gen. Microbiol. 129, 1743-1813).

Growth, colonial appearance, substrate and aerial mycelium colour andpigment production for strain Microbispora sp. ATCC PTA-5024 arerecorded in Table I. Vegetative growth is present on most of the mediaused, differently from the aerial mycelium that is present only on someof them. No evident pigmentation is shown on any medium used.Physiological characteristics of the strain are presented in Table II.Growth and aerial mycelium production are present at 17° C. but not at43° C. Production of aerial mycelium on ISP2 is present at pH higherthan 6, while it is absent in presence of 1% NaCl.

The ability to use various carbohydrates for growth is shown in TableIII.

TABLE I growth characteristics of Microbispora sp. ATCC PTA-5024 REVERSECOLOUR MEDIUM GROWTH & MORPHOLOGY CODE ISP 2 Abundant growth, wrinkledsurface; 5 E 12 Yeast good production of pinkish (2A8) orangish/redextract- aerial mycelium. Malt Slight production of extractorangish/light brown soluble agar pigment. ISP 3 Abundant growth; goodproduction 11 H 10 Oatmeal of pinkish (2A8) aerial mycelium,orangish/pink agar particularly on the arms of the cross-hatchedstreakes. Slight production of orangish soluble pigment. ISP 4 Goodgrowth; no aerial mycelium 11 I 9 Inorganic produced. orange salts- Nosoluble pigments produced. Starch Starch hydrolysed. agar Glu/AspDiscrete growth, thin; production 12 K 12 Glucose- of thin,beige/pinkish (9B4) orangish/light- Asparagine aerial mycelium on thearms of the brown agar cross-hatched streakes. No soluble pigmentsproduced. ISP 6 Scant growth, with pinkish single nd Peptone- coloniesgrown in height, yeast convolute, with a smooth surface; extract- noaerial mycelium produced. No iron agar darkening of the medium. ISP 7Poor growth of a thin, nd Tyrosine orangish/light-brown substrate agarmycelium; no aerial mycelium produced. No darkening of the medium.ISP3 + YE Abundant growth, wrinkled surface; 4 B 12 Oatmeal/ very scantproduction of thin, orangish/red 1% yeast pinkish aerial mycelium.extract No soluble pigments produced. agar(ISP4 and Glucose-Asparagine Agar Added with 1 ml/L of VitaminsSolution)

TABLE II physiological characteristics of Microbispora sp. ATCCPTA-5024. TEST REACTION Starch hydrolysis Positive Casein hydrolysisNegative Calcium malate digestion Negative Litmus milk peptonizationNegative Litmus milk coagulation Negative Gelatin liquefaction Negativeto slightly positive Tyrosine reaction Negative Nitrate reductionPositive PH range of growth (14 days) no growth at 4.2, good at 5.5 to8.8; not tested out of this range. Aerial mycelium absent at pH ≦ 6.5NaCl % tolerance ≦2; absence of aerial mycelium at ≧1. Temperature rangeof growth 17° C. to 37° C. Presence of aerial mycelium in the wholerange; no growth at 43° C.

TABLE III utilization of carbon sources by Microbispora sp. GrowthCarbon source (14 days) Arabinose ++ Cellulose − Fructose ++ Inositol+/− Mannitol +++ Raffinose − Rhamnose − Sucrose +++ Xylose +++ Glucose++ Glycerol ++ No sugar − +++ abundant; ++ good growth; + moderategrowth; +/− scant growth; − no growth; aerial mycelium always absent.Chemotaxonomical Characteristics of Microbispora sp. ATCC PTA-5024

Microbispora sp. ATCC PTA-5024 was grown in GYM medium (glucose 4 g/l;yeast extract 4 g/l; malt extract 10 g/l) at 28° C. on a rotary shakerand the mycelium harvested, washed twice with sterile distilled waterand subsequently freeze-dried. Analyses of amino acids were carried outaccording to the method of Staneck and Roberts, (J. L. Staneck and G. D.Roberts, (1974): “Simplified approach to identification of aerobicactinomycetes by thin-layer chromatography”, Appl. Microbiol. 28:226-231). Menaquinones and polar lipids were extracted following theprocedure of Minnikin et al. (D. E. Minnikin, A. G. O'Donnell, M.Goodfellow, G. Alderson, M. Athalye, A. Schaal and J. H. Parlett,(1984): “An integrated procedure of isoprenoid quinones and polarlipids”, J. Microbiol. Meth. 2: 233-241). Polar lipids were analysed bythin layer chromatography (D. E. Minnikin, V. Patel, L. Alshamaony, andM. Goodfellow, (1977): “Polar lipid composition in the classification ofNocardia and related bacteria”, Int. J. Syst. Bacteriol. 27:104-117),menaquinones by HPLC (R. M. Kroppenstedt, (1982): “Separation ofbacterial menaquinones by HPLC using reverse phase RP18 and a silverloaded ion exchanger as stationary phase”, J. Liquid. Chromat.5:2359-2367; R. M. Kroppenstedt, (1985): “Fatty acid and menaquinoneanalysis of actinomycetes and related organisms”, in: Chemical Methodsin Bacterial Systematics. No 20 SAB Technical Series pp. 173-199, M.Goodfellow and D. E. Minnikin eds, Academic Press, London) and fattyacid methyl esters by gas-liquid chromatography respectively (L. T.Miller, (1982): “A single derivatization method for bacterial fatty acidmethyl esters including hydroxy acids”, J. Clin. Microbiol. 16: 584-586;M. Sasser, (1990): “Identification of bacteria by gas chromatography ofcellular fatty acids”, USFCC News Letters 20:1-6). The presence ofmycolic acids was checked by the method of Minnikin et al. (D. E.Minnikin, L. Alshamaony, and M. Goodfellow, (1975): “Differentiation ofMycobacterium, Nocardia and related taxa by thin layer chromatographicanalysis of whole organism methanolyzates”, J. Gen. Microbiol. 88:200-204).

Whole cell hydrolyzates of strain Microbispora sp. ATCC PTA-5024 containmeso-diaminopimelic acid as the diammino acid of the peptidoglycan. Thepredominant menaquinones are MK-9(III, VIII-H₄), MK-9(H₂) and MK-9(H₀).The polar lipid pattern is characterized by the presence ofphosphatidylethanolamine, methylphosphatidylethanolamine,phosphatidyl-glycerol, diphosphatidyl-glycerol, phosphatidyl-inositol,phosphatidyl-inositolmannosides and N-acetylglucosamine containingphospolipd, i.e. phospholipid type IV according to Lechevalier et al.(H. A. Lechevalier, C. De Briève and M. P. Lechevalier, (1977):“Chemotaxonomy of aerobic actinomycetes: phospholipid composition”,Biochem. Syst. Ecol. 5: 246-260). The major components of fatty acidpattern are anteiso 15:0, iso 16:0, n-16:0, anteiso 17:0, and10-methyl-heptadecanoic (10-Me-17:0), i.e 3c sensu Kroppenstedt (R. M.Kroppenstedt, (1985): “Fatty acid and menaquinone analysis ofactinomycetes and related organisms”, in: Chemical Methods in BacterialSystematics. No 20 SAB Technical Series pp. 173-199. M. Goodfellow andD. E. Minnikin eds, Academic Press, London). Mycolic acids are notdetected.

Microbispora sp. ATCC PTA-5024 16S rDNA Sequencing

The partial sequence of the 16 rRNA gene (16S rDNA), i.e 1443nucleotides, corresponding to 95% of the entire rRNA, of strainMicrobispora sp. ATCC PTA-5024, was achieved following publishedprocedures (P. Mazza, P. Monciardini, L. Cavaletti, M. Sosio and S.Donadio, (2003): “Diversity of Actinoplanes and related genera isolatedfrom an Italian soil”, Microbial Ecol. 5:362-372). It is reported in SEQID NO 1.

This sequence was compared with that of strain Microbispora corallinaNRRL 30420 (MF-BA-1768), as reported in U.S. Pat. No. 6,551,591 B1. Thetwo sequences were aligned and differences were found at 31 out of 1456aligned positions, accounting for an overall sequence divergence of2.13%. Any two strains sharing less than 97.5% sequence identity usuallybelong to different species (Stackebrandt, E. and Embley, M. T. (2000)“Diversity of Uncultered Microorganisms in the Environment”. In:Nonculturable Microorganisms in the Environment, R. R. Colwell and D. J.Grimes (eds). ASM, Press, Washington D.C., pp. 57-75). Therefore a 2%level of sequence divergence is quite high (Rossellò-Mora, R., andAmann, R. (2001). “The Species Concept for Prokaryotes”. FEMS Microbiol.Rev. 25: 39-67) and indicates that Microbispora sp. ATCC PTA-5024 andMicrobispora corallina NRRL 30420 (MF-BA-1768) are different strains.

Identity of Strain Microbispora sp. ATCC PTA-5024

The strain producing antibiotic 107891 is assigned to the genusMicrobispora, family Streptosporangiaceae because of the followingchemotaxonomical and morphological characteristics:

-   -   presence of meso-diaminopimelic acid in the cell wall;    -   major amount of MK-9(III, VIII-H₄) and phospholipid type IV        according to Lechevalier et al. (H. A. Lechevalier, C. De Briève        and M. P. Lechevalier, (1977): “Chemotaxonomy of aerobic        actinomycetes: phospholipid composition”, Biochem. Syst. Ecol.        5: 246-260);    -   fatty acid profile of 3c sensu Kroppenstedt (R. M. Kroppenstedt,        (1992): “The genus Nocardiopsis”, in: The Prokariotes, Vol II,        pp. 1139-1156, A. Balows, H. Truper, M. Dworkin, W. Harder        and K. H. Schleifer eds; New York, Springer-Verlag);    -   absence of mycolic acids;    -   formation of characteristic longitudinal pairs of spores on the        tips of short sporophores laterally branching from aerial        hyphae. Non-motile spores.    -   partial sequence of the 16 rRNA gene (16S rDNA), i.e 1443        nucleotides, corresponding to 95% of the entire rRNA, reported        in SEQ ID NO.1, showing >97% identity to 16S rDNA sequences from        described Microbispora species.

As with other microorganisms, the characteristics of strain producingantibiotic 107891 are subject to variation. For example, artificialvariants and mutants of the strain can be obtained by treatment withvarious known mutagens, such as U.V. rays, and chemicals such as nitrousacid, N-methyl-N′-nitro-N-nitrosoguanidine, and many others. All naturaland artificial variants and mutants of strain Microbispora sp. ATCCPTA-5024 capable of producing antibiotic 107891 are deemed equivalent toit for the purpose of this invention and therefore within the scope ofinvention.

Extraction and Purification of Antibiotic 107891

As mentioned above, antibiotic 107891 is found almost equallydistributed both in the mycelium and in the filtered fraction of thefermentation broth.

The harvested broth may be processed to separate the mycelium from thesupernatant of the fermentation broth and the mycelium may be extractedwith a water-miscible solvent to obtain a solution containing the 107891antibiotic, after removal of the spent mycelium. This mycelium extractmay then be processed separately or in pool with the supernatantaccording to the procedures reported hereafter for the supernatantfraction. When the water-miscible solvent may cause interferences withthe operations for recovering the antibiotic from the mycelium extract,the water-miscible solvent may be removed by distillation or may bediluted with water to a non-interfering concentration.

The term “water-miscible solvent” as used in this application, isintended to have the meaning currently given in the art of this term andrefers to solvents that, at the conditions of use, are miscible withwater in a reasonably wide concentration range. Examples ofwater-miscible organic solvents that can be used in the extraction ofthe compounds of the invention are: lower alkanols, e.g. (C₁-C₃)alkanols such as methanol, ethanol, and propanol; phenyl (C₁-C₃)alkanols such as benzyl alcohol; lower ketones, e.g. (C₃-C₄) ketonessuch as acetone and ethyl methyl ketone; cyclic ethers such as dioxaneand tetrahydrofuran; glycols and their products of partialetherification such as ethylene glycol, propylene glycol, and ethyleneglycol monomethyl ether, lower amides such as dimethylformamide anddiethylformamide; acetic acid dimethylsulfoxide and acetonitrile.

The recovery of the compound from the supernatant of the fermentationbroth of the producing microorganism is conducted according to known perse techniques which include extraction with solvents, precipitation byadding non-solvents or by changing the pH of the solution, by partitionchromatography, reverse phase partition chromatography, ion exchangechromatography, molecular exclusion chromatography and the like or acombination of two or more of said techniques. A procedure forrecovering the compounds of the invention from the filtered fermentationbroth includes extraction of antibiotic 107891 with water-immiscibleorganic solvents, followed by precipitation from the concentratedextracts, possibly by adding a precipitating agent.

Also in this case, the term “water-immiscible solvent” as used in thisapplication, is intended to have the meaning currently given in the artto said term and refers to solvents that, at the conditions of use, areslightly miscible or practically immiscible with water in a reasonablywide concentration range, suitable for the intended use.

Examples of water-immiscible organic solvents that can be used in theextraction of the compounds of the invention from the fermentation brothare:

alkanols of at least four carbon atoms which may be linear, branched orcyclic such as n-butanol, 1-pentanol, 2-pentanol, 3-pentanol, 1-hexanol,2-hexanol, 3-hexanol, 3,3-dimethyl-1-butanol, 4-methyl-1-pentanol,3-methyl-1-pentanol, 2,2-dimethyl-3-pentanol, 2,4-dimethyl-3-pentanol,4,4-dimethyl-2-pentanol, 5-methyl-2-hexanol, 1-heptanol, 2-heptanol,5-methyl-1-hexanol, 2-ethyl-1-hexanol, 2-methyl-3-hexanol, 1-octanol,2-octanol, cyclopentanol, 2-cyclopentylethanol,3-cyclopenthyl-1-propanol, cyclohexanol, cycloheptanol, cyclooctanol,2,3-dimethyl-cyclohexanol, 4-ethylcyclohexanol, cyclooctylmethanol,6-methyl-5-hepten-2-ol, 1-nonanol, 2-nonanol, 1-decanol, 2-decanol, and3-decanol; ketones of at least five carbon atoms such asmethylisopropylketone, methylisobutylketone, methyl-n-amylketone,methylisoamylketone and mixtures thereof.

As known in the art, product extraction from the filtered fermentationbroth may be improved by adjusting the pH at an appropriate value,and/or by adding a proper organic salt forming an ion pair with theantibiotic, which is soluble in the extraction solvent.

As known in the art, phase separation may be improved by salting theaqueous phase.

When, following an extraction, an organic phase is recovered containinga substantial amount of water, it may be convenient to azeotropicallydistill water from it. Generally, this requires adding a solvent capableof forming minimum azeotropic mixtures with water, followed by theaddition of a precipitating agent to precipitate the desired product, ifnecessary. Representative examples of organic solvents capable offorming minimum azeotropic mixtures with water are: n-butanol, benzene,toluene, butyl ether, carbon tetrachloride, chloroform, cyclohexane,2,5-dimethylfuran, hexane, and m-xylene; the preferred solvent beingn-butanol.

Examples of precipitating agents are petroleum ether, lower alkylethers, such as ethyl ether, propyl ether, and butyl ether, and loweralkyl ketones such as acetone.

According to a preferred procedure for recovering antibiotic 107891, thefiltered fermentation broth can be contacted with an adsorption matrixfollowed by elution with a polar, water-miscible solvent or a mixturethereof, concentration to an oily residue under reduced pressure, andprecipitation with a precipitating agent of the type already mentionedabove.

Examples of adsorption matrixes that can be conveniently used in therecovery of the compounds of the invention, are polystyrene or mixedpolystyrene-divinylbenzene resins (e.g. M112 or S112, Dow Chemical Co.;Amberlite® XAD2 or XAD4, Rohm & Haas; Diaion HP 20, Mitsubishi), acrylicresins (e.g. XAD7 or XAD8, Rohm & Haas), polyamides such aspolycaprolactames, nylons and cross-linked polyvinylpyrrolidones (e.g.Polyamide-CC 6, Polyamide-SC 6, Polyamide-CC 6.6, Polyamide-CC 6AC andPolyamide-SC 6AC, Macherey-Nagel & Co., Germany; PA 400, M. Woelm AG,Germany); and the polyvinylpirrolidone resin PVP-CL, (Aldrich ChemieGmbH & Co., KG, Germany) and controlled pore cross-linked dextrans (e.g.Sephadex® LH-20, Pharmacia Fine Chemicals, AB). Preferably, polystyreneresins are employed, particularly preferred being the Diaion HP 20resin.

In the case of polystyrene resins, polystyrene-divinylbenzene resins,polyamide resins or acrylic resins a preferred eluent is awater-miscible solvent or its aqueous mixtures. The aqueous mixtures cancontain buffers at appropriate pH value.

Also in this case, the term “water-miscible solvent”, as used in thisdescription and claims, is intended to have the meaning currently givenin the art to said term as described above.

The successive procedures for the isolation and purification of theantibiotic may be carried out on the pooled extracts from the brothsupernatant and from the mycelium. For example, when the portion of theantibiotic product contained in the filtered fermentation broth orsupernatant is recovered by absorption on an absorption resin and theportion of the antibiotic product contained in the mycelium is extractedtherefrom with a water-miscible solvent, followed by adsorption onto anabsorption resin, the eluted fractions from each of the two sets ofabsorption resins may be combined, optionally after concentration, andthen further processed as a unitary crop. Alternatively, when the twosets of absorption resins utilized for the separate extraction stagesare of the same type and have the same functional characteristics, theymay be pooled together and the mixture may be submitted to a unitaryelution step, for instance, with a water-miscible solvent or a mixturethereof with water.

In any case, whatever may be the procedure adopted for recovering thecrude antibiotic 107981, the successive purification step is usuallycarried out on the mixture of the crude materials resulting from thecombination of the products originating from the separate extractionstages.

Purification of the crude antibiotic 107891, can be accomplished by anyof the known per se techniques but is preferably conducted by means ofchromatographic procedures. Examples of these chromatographic proceduresare those reported in relation to the recovery step and include alsochromatography on stationary phases such as silica gel, alumina,activated magnesium silicate an the like or reverse phasechromathography on silanized silica gel having various functionalderivatizations, and eluting with water miscible solvents or aqueousmixture of water-miscible solvents of the kind mentioned above.

For instance, preparative HPLC chromatography may be employed, usingRP-8 or RP-18 as stationary phase and a mixture of HCOONH₄ buffer: CH₃CNas eluting system.

The active fractions recovered from the purification step are pooledtogether, concentrated under vacuum, precipitated by addition of aprecipitating agent of the kind mentioned above and dried or lyophilisedin single or iterative rounds. In the case the product contains residualamounts of ammonium formate or other buffering salts, these may beremoved by absorption of the antibiotic 107891 on solid phase extractioncolumn, for instance a reverse phase resin column such as SPE SupercleanLCP18 Supelco (Bellefonte Pa., USA) followed by washing with distilledwater and elution with an appropriate aqueous solvent mixture, e.g.,ethanol:water. The antibiotic is then recovered by removing the elutionsolvents.

Accordingly, a purified antibiotic 107891 complex dried preparation isobtained as a white powder.

As usual in this art, the production as well as the recovery andpurification steps may be monitored by a variety of analyticalprocedures including inhibitory assay against susceptible microorganismsand analytical control using the HPLC or HPLC coupled with massspectrometry.

A preferred analytical HPLC technique is performed on a Watersinstrument (Waters Chromathography, Milford, Mass.) equipped with acolumn Waters Simmetry-shield RP8, 5μ (250×4.6 mm) eluted at 1 ml/minflow rate and at 50° C. temperature.

Elution was with a multistep program: Time=0 (30% phase B); Time=8 min(30% Phase B); Time=28 min (40% of phase B).

Phase A was acetonitrile: 100 mM ammonium formate buffer (pH:5.0) 5:95(v/v) and Phase B was acetonitrile. UV detector was at 282 nm.

The effluent from the column was splitted in a ratio 5:95 and themajority (ca. 950 μl/min) was diverted to photodiode array detector. Theremaining 50 μl/min were diverted to the ESI interface of a Finnigan LCQion trap mass spectrometer (Thermoquest, Finnigan MAT, San Josè Calif.).

The mass spectrometric analysis was performed under the followingconditions:

Sample Inlet Conditions:

Sheat gas (N₂) 60 psi;

Aux gas (N₂) 5 psi;

Capillary heater 250° C.;

Sample Inlet Voltage Settings:

Polarity both positive and negative;

Ion spray voltage +/−5 kV;

Capillary voltage +/−19V;

Scan conditions: Maximum ion time 200 ms;

Ion time 5 ms;

Full micro scan 3;

Segment: duration 30 min, scan events positive (150-2000 m/z) andnegative (150-2000 m/z).

In these analytical HPLC conditions the antibiotic 107891 Factors A1 andA2 showed retention times of 13.2 min and 13.9 min, respectively. In thesame HPLC system Ramoplanin A2 Factor (L. Gastaldo, R. Ciabatti, F.Assi, E. Restelli, J. K. Kettenring, L. F. Zerilli, G. Romanò, M. Denaroand B. Cavalleri, (1992): “Isolation, structure determination andbiological activity of A-16686 Factors A′1, A′2 and A′3glycolipodepsipeptide antibiotics”, J. Ind. Microbiol. 11: 13-18) elutedwith a retention time of 7.5 min.

Antibiotic 107891 Factors A1 e A2 may be separated from a purifiedsample of antibiotic 107891 complex by means of preparative HPLC.

Factor A1 was separated and purified on a Symmetry Prep. C18 column fromthe purified antibiotic 107891 complex dissolved in DMSO: formic acid95:5 (v/v) using a 25 minutes linear gradient elution from 30% to 45% ofphase B at 3.5 ml flow rate.

Phase B was acetonitrile. Phase A was 25 mM ammonium formate buffer pH4.5: acetonitrile 95:5 (v/v). The eluted fractions containing pureantibiotic 107891 Factor A1 were pooled and concentrated under vacuum.The residual solution was lyophilised yielding pure Factor A1 as a whitepowder.

Factor A2 was separated and purified by isocratic elution on a SymmetryPrep. C18 column from a sample of purified antibiotic 107891 complexdissolved in acetic acid:acetonitrile: 100 mM ammonium formate buffer(pH 4) 50:120:80 (v/v) mixture. Isocratic elution was performed at a 7ml flow rate with a mixture 100 mM ammonium formate buffer pH 4:acetonitrile in the proportion 82.5:17.5 (v/v). The eluted fractionscontaining pure antibiotic 107891 Factor A2 were pooled and concentratedunder vacuum. The residual solution was liophilized yielding pure FactorA2 as a white powder.

Since antibiotic 107891 and its Factors A1 and A2, as shown by acid/basetitration in 2-methoxyethanol (MCS):H₂O 12:3 (v/v), contains a basicfunction, they are capable of forming salts with suitable acidsaccording to conventional procedures and they may exist also in the freebase form.

Antibiotic 107891 and its Factors A1 and A2, when obtained in the freebase form, may be converted with acids into the corresponding salts,which include non-toxic pharmaceutically acceptable salts. Suitablesalts include those salts formed by standard reaction with both organicand inorganic acids such as, for example, hydrochloric, hydrobromic,sulfuric, phosphoric, acetic, trifluoroacetic, trichloroacetic,succinic, citric, ascorbic, lactic, maleic, fumaric, palmitic, cholic,pamoic, mucic, glutamic, camphoric, glutaric, glycolic, phthalic,tartaric, lauric, stearic, salicylic, methanesulfonic, benzenesulfonic,sorbic, picric, benzoic, cinnamic and the like acids. The addition saltsof antibiotic 107891 and its Factors A1 and A2, with acids can beprepared according to the usual procedures commonly employed. As anexample, antibiotic 107891 or its Factor A1 or its Factor A2, in thefree base form, is dissolved into the minimum amount of a suitablesolvent, typically a lower alkanol, or a lower alkanol/water mixture,the stoichiometric amount of a suitable selected acid is gradually addedto the obtained solution and the obtained salt is precipitated by theaddition of a non-solvent. The addition salt which forms is thenrecovered by filtration or evaporation of the solvents.

Alternatively, these salts can be prepared in a substantially anhydrousform through lyophilization; in this case a salt of antibiotic 107891 orits Factor A1 or its Factor A2 with volatile acid is dissolved with asuitable amount of non-volatile acid. The solution is then filtered fromany insolubles and is lyophilized in single or iterative rounds.

A specific addition salt may be also obtained from a solution of anothersalt form of antibiotic 107891 or its Factor A1 or its Factor A2 whenthe desired salt precipitates upon addition of the appropriate anion.

The transformation of the non salts compound of the invention into thecorresponding addition salts, and the reverse, i.e. transformation of anaddition salt of a compound of the invention into the non-salt form arewithin the ordinary technical skill and are encompassed by the presentinvention.

The formation of salts of antibiotic 107891 and its Factors A1 and A2may serve several purposes, including the separation, purification ofsaid antibiotic 107891 and its Factors A1 and A2 and their use astherapeutical agents or animal growth promoters. For therapeuticalpurposes, the pharmaceutically acceptable salts are usually employed.

The term “pharmaceutically acceptable salts” identifies those non-toxicsalts which can be utilized in the therapy of warm-blooded animals.

The antibiotic 107981 complex, its Factors A1 and A2 and a mixture ofsaid Factors in any proportion can be administered as such or inmixtures with pharmaceutically acceptable carriers and can also beadministered in conjunction with other antimicrobial agents such aspenicillins, cephalosporins, aminoglycosides and glycopeptides.

Conjunctive therapy, thus includes sequential, simultaneous and separateadministration of the active compound in a way that the therapeuticeffects of the first administered one is not entirely disappeared whenthe subsequent is administered.

The compounds of the invention, or its pharmaceutically acceptableaddition salts, can be formulated into forms suitable for parenteral,oral or topical administration. For iv. administration in the treatmentof any infection involving a microorganism susceptible to theantibiotic, a parenteral formulation is, for instance, in water with anappropriate solubilising agent such as polypropylene glycol ordimethylacetamide and a surface-active agent (e.g. polyoxyethylenesorbitan mono-oleate or polyethoxylated castor oil) or cyclodextrins orphospholipid based formulations in sterile water for injection. Aninjectable formulation may be also obtained with an appropriatecyclodextrin.

The antibiotic 107981 complex, its Factors A1 and A2 and a mixture ofsaid Factors in any proportion may also be used in a suitablepharmaceutical form such as a capsule, a tablet or an aqueous suspensionfor oral administration or with conventional creams or jellies fortopical applications. Besides their use as medicaments in human andveterinary therapy, the compounds of the invention can also be used asanimal growth promoters. For this purpose, a compound of the inventionis administered orally in a suitable feed. The exact concentrationemployed is that which is required to provide for the active agent in agrowth promotant effective amount when normal amounts of feed areconsumed.

The addition of the active compound of the invention to animal feed ispreferably accomplished by preparing an appropriate feed premixcontaining the active compound in an effective amount and incorporatingthe premix into the complete ration. Alternatively, an intermediateconcentrate or feed supplement containing the active ingredient can beblended into the feed. The way in which such feed premixes and completerations can be prepared and administered are described in referencebooks (such as “Applied Animal Nutrition”, W.H. Freedman and CO., S.Francisco, U.S.A., 1969 or “Livestock Feeds and Feeding” 0 and B books,Corvallis, Ore., U.S.A., 1977).

Physico-Chemical Characteristics of Antibiotic 107891

A) Mass Spectrometry: in MS experiments on a Thermofinnigan LCQ decainstrument fitted with an electrospray source, using Thermofinnigancalibration mix, antibiotic 107891 gives two doubly protonated ions atm/z=1124 and at m/z 1116 corresponding to lowest isotope composition ofthe complex Factors A1 and A2, respectively. The electrospray conditionswere: Spray Voltage: 4.7 kV; Capillary temperature: 220° C.; CapillaryVoltage: 3 V; Infusion mode 10 μl/min. Spectra were recorded from a 0.2mg/ml solution in methanol/water 80/20 (v/v) with trifluoracetic acid0.1% and are reported in FIG. 1A (full scan low resolution spectrum) and1B (zoom-scan high resolution spectrum).

B) The infrared spectrum of antibiotic 107891 recorded in KBr with aBruker FT-IR spectophotometer model IFS 48, exhibits absorption maximaat (cm⁻¹): 3263; 2929; 1661; 1533; 1402; 1114; 1026. Infrared spectrumis reported in FIG. 2. Absorption bands at 1631, 1596 and 1346 areattributed to residual amounts of ammonium formate.

C) The U.V. spectrum of antibiotic 107891, performed in methanol/H₂O (inratio 80:20) with a Perkin-Elmer spectrophotometer Lambda 16, exhibitstwo shoulders at 226 and 267 nm. UV spectrum is reported in FIG. 3

D) ¹H-NMR spectrum was recorded in the mixture methanol-d4:H₂O (pH 4.3HCl) 40:10 (v/v) at 40° C. on a Bruker AMX 600 spectrometer applying awater suppression sequence. As internal standard the residual signal ofmethanol-d4 at 3.31 ppm was considered.

The ¹H-NMR spectrum of antibiotic 107891 is reported in FIG. 4. ¹H NMRspectrum of antibiotic 107891 dissolved in methanol-d4:H₂O (0.01N HCl)40:10 (v/v) exhibits the following groups of signals (in ppm) at 600 MHzusing MeOH-d4 as internal standard (3.31 ppm), [δ=ppm, multiplicity;(attribution)]: 0.93 d (CH₃), 0.98 d (CH₃), 1.07 t (overlapped CH₃'s),1.18 t (overlapped CH₃'s), 1.26 s (CH₃), 1.30 t (overlapped CH₃'s),1.62-1.74 m (CH₂), 1.78 d (CH₃), 1.80 d (CH₃), 2.03 m (CH₂), 2.24 m(CH), 2.36 m (CH₂), 2.72-3.8 m (peptidic alpha CH's), 3.8-5.2 m(peptidic alpha CH's), 5.53-6.08 s (CH₂), 5.62 d (CH double bond), 6.42m (CH), 6.92 d (CH double bond), 7.0-7.55 m (aromatic CH's), 7.62-10.4 dand m (aromatic and peptidic NH's).

E) ¹³C-NMR spectrum was recorded in the mixture methanol-d4:H₂O (pH 4.3HCl) 40:10 (v/v) at 40° C. on a Bruker AMX 600 spectrometer using asinternal standard the residual signal of methanol-d4 at 49.15 ppm. The¹³C-NMR spectrum bb decoupled of antibiotic 107891 is reported in FIG.5.

¹³C NMR spectrum of antibiotic 107891 dissolved in methanol-d4:H₂O (0.01N HCl) 40:10 (v/v) exhibits the following groups of signals (in ppm) at600 MHz using MeOH-d4 as internal standard (49.15 ppm), [δ=ppm;(attribution)]: 13.6-23.2 (aliphatic CH₃'s), 26.16-73 (aliphatic CH₂'sand peptidic alpha CH's), 105-136 (aromatic and double bonds CH's andquaternary carbons), 164.3-176.3 (peptidic carbonyls).

F) Antibiotic 107891 complex was dissolved in 2-methoxyethanol (MCS):H₂O12:3 (v/v) containing a molar excess of 0.01 M hydrochloric acid. Thesolution was then back titrated with a solution of 0.01 N potassiumhydroxide. The resulting titration curve showed one basic ionizablefunction.

Amino Acids Composition of Antibiotic 107891 and its Factors A1 and A2

A) Determination of “Acid Resistant” Aminoacids in Antibiotic 107891Complex

Antibiotic 107891 was submitted to complete acidic hydrolysis (HCl 6N,105° C., 24 h) and amino acid components of the antibiotic resistant toacid treatment were identified. Acid labile amino acids are notdetectable with this approach. The hydrolysate was studied by HPLC-MSand GC-MS analysis, after suitable derivatization, in comparison with amixture of standard amino acids similarly derivatized. For HPLC analysisthe hydrolyzed sample was treated with6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AccQ-Tag™ Fluor reagentkit), for GC anlysis with a mixture of 3N HCl in anhydrous methanol andtrifluoroacetic anhydride.

The qualitative HPLC analysis was carried out on a liquid chromatographysystem with simultaneous DAD and MS detection. The HPLC method had thefollowing conditions:

Column: AccQ-Tag™ (Waters C18 NovoPak 4 μm 3.9×150 mm)

Column temperature: 37° C.

Flow: 1 mL/min.

Phase A: Ammonium acetate 140 mM pH 5 (acetic acid)

Phase B: Water:acetonitrile 60:40 (v/v)

Elution Program

Time (min.) 0 5 30 35 40 41 % B 5 5 80 95 95 5UV detection: 254 nmMS conditions were the following:Spectrometer: Finnigan LCQ Deca equipped with standard electrospraysource.Capillary temperature: 250° C.Source voltage: 4.70 KVSource current: 80 μACapillary voltage: −15V

The qualitative GC analysis was carried out on a gas cromatographerfitted with MS-EI detection.

The GC method had the following conditions:

Column: J & W Scientific DB-5, 30 m×0.254 mm ID×0.25 μm FT

Carrier gas: helium

Injection mode: splitless

Injector temperature: 200° C.

Transfer line temperature: 300° C.

Temperature program: from 50° C. to 100° C. at 2.5° C./min (10 min),from 100° C. to 250° C. at 10° C./min (15 min), 15 min at 250° C.

Injection volume: 1 μl

MS conditions were the following:

Spectrometr: Finnigan TSQ700

Ionisation mode: Electron impact

Voltage Setting:

Filament current: 400 mA

Electron multiplier: 1400 V

Electron energy: 70 eV

Positive Ion Mode

Scan Condition:

Scan range: 40-650 amu

Scan time: 1 sec

In the LC/MS and GC/MS chromatograms obtained on the hydrolysate ofantibiotic 107891, the following amino acids were identified along withother unidentified peaks: lanthionine, methyllanthionine, glycine,proline, valine, aspartic acid (NMR studies indicate that this is atransformation product of asparagine, which generates aspartic acid byhydrolysis), phenylalanine and leucine.

Antibiotic 107891 Factors A1 and A2 were submitted to complete acidichydrolysis in the same conditions (derivatization and HPLC-MS) reportedfor the complex. The GC-MS analysis was carried out on a Thermo FinniganTrace GC-MS instrument equipped with PTV injector

The GC method had the following conditions:

Column: Restek RTX-5MS, 15 m×0.25 mm ID×0.25 μm FT

Carrier gas: helium

Interface temperature: 250° C.

Temperature program: 1.5 min at 50° C., from 50° C. to 100° C. at 20°C./min, 1 min at 100° C., from 100° C. to 135° C. at 20° C./min, 1 minat 135° C., from 135° C. to 250° at 20° C./min, 1 min at 250° C.

Injection volume: 1 μl

Injector: splitless mode, base temperature 50° C., transfer temperature280° C., transfer rate 14.5° C./min

MS conditions were the following:

Ionisation mode: Electron impact

Voltage Setting:

Filament current: 149 μA

Electron multiplier: 200 V

Electron energy: 70 eV

Positive Ion Mode:

Scan Condition:

Scan range: 33-500 amu

Scan time: 0.6 sec

In the hydrolysate of Factor A1 of antibiotic 107891, HPLC/MS and GC/MSchromatograms showed the presence of the following amino acids alongwith other unidentified peaks: lanthionine, methyllanthionine, glycine,proline, valine, aspartic acid (NMR studies indicate that this is atransformation product of asparagine, which generates aspartic acid byhydrolysis), phenylalanine and leucine.

The above procedure carried out on Factor A2 revealed the presence ofthe following amino acids along with other unidentified peaks:lanthionine, methyllanthionine, glycine, proline, valine, aspartic acid(NMR studies indicate that this is a transformation product ofasparagine, which generates aspartic acid by hydrolysis), phenylalanineand leucine.

B) Determination of 5-Chlorotyptophan in Antibiotic 107891 Complex andin its Factor A1 and Factor A2.

Complete hydrolysis of purified 107891 complex and its single Factors A1and A2 was performed according to the method described by Simpson R J,Neuberger M R, Liu T Y, “Complete Aminoacid Analysis of Proteins from aSingle Hydrolysate”. Journal Biol. Chem (United States), Apr. 10, 1976,251 (7), 1936-40.

This hydrolysis procedure prevents degradation of amino acids normallyunstable during mineral acid digestion and thus allows the determinationof these amino acids, including tryptophan, from a hydrolysate of apeptide. A standard sample of 5-chloro-DL-tryptophan was purchased fromBiosynt AG, Staad, Switzerland and its structure was confirmed by NMRanalysis; DL-tryptophan was purchased from Merck KGaA, Darmstadt,Germany.

Factor A1 (1.5 mg) was suspended in 0.6 ml of 4N methanesulfonic acidcontaining 0.2% (w/v) 3-(2-aminoethyl)indole as catalyst for thehydrolysis. The hydrolysis was carried out at 115° C. for 16 hours. Thehydrolysate was then neutralized with 5N NaOH and diluted with an equalamount of distilled water. 100 μl of this solution was analysed byLC-MS. The separation was performed on a Symmetry C₁₈ (5 μm) 4.6×250 mm.column (Waters Co. Milford Mass., USA) equipped with a Symmetry C₁₈ (5μm) 3.9×20 mm precolumn. Elution was performed at 1 ml/min flow ratewith a 25 min. linear gradient from 0% to 50% of Phase B. Phase A was 25mM HCOONH₄ buffer pH 4.5:CH₃CN 95:5 (v/v) and Phase B was CH₃CN. UVdetection was at 280 nm. The HPLC equipment was coupled with a FinniganLCQ ion trap Mass Spectrometer (Thermoquest, Finnigan MAT, San Josè, CA,USA). 50 μl/min of the effluents from the column were diverted to theElectrospray Ionization (ESI) interface of the LCQ mass spectrometer.The MS analysis was performed under the following conditions: sampleinlet: shear gas (N₂) 60 psi; capillary heater 210° C.; sample inletvoltage polarity: both positive and negative; ion spray voltage +/−4.5KV; capillary voltage +/−21 V; scan conditions: maximum ion time 50 ms;full micro: scan 3.

Standards of tryptophan and 5-chlorotryptophan eluted at retention timesof 8.1 minutes and 11.5 minutes corresponding to a M+H⁺ at m/z 205 and239, respectively. In the hydrolysate of antibiotic 107891 Factor A1 thepresence of a peak at 11.5 minutes with m/z at 238.97 indicated thepresence of 5-chlorotryptophan.

Standard tryptophan was detectable with the chromatographic system usedwith a detection limit of 0.3 μg/ml. This value is lower than the valuewhich would have been indicative of the presence of said aminoacid inthe tested antibiotic sample. No tryptophan was detected within theabove said limit in the chromatogram of the hydrolysate of antibiotic107891 Factor A1. Identical results were obtained from LC-MS analysis ofa hydrolysate of Factor A2 and of a hydrolysate of a purified sample ofantibiotic 107891 complex.

Mass Spectrometry of Antibiotc 107891 Factor A1 and Factor A2

Antibiotic 107891 Factor A1 gives a doubly protonated ion at m/z=1124and Factor A2 at m/z 1116 corresponding to the lowest isotopecomposition in MS experiments on a Thermofinnigan LCQ deca instrumentfitted with an electrospray source, using Thermofinnigan calibrationmix. The electrospray conditions were: Spray Voltage: 4.7 kV; Capillarytemperature: 250° C.; Capillary Voltage: 8 V; Infusion mode 10 μl/min.Spectra were recorded from a 0.1 mg/ml solution in acetonitrile:water50:50 (v/v) with acetic acid 0.5% and are reported in FIG. 6A (full scanlow resolution spectrum) and 6B (zoom-scan high resolution spectrum) andin FIG. 7A (full scan low resolution spectrum) and B (zoom-scan highresolution spectrum).

The exact mass of antibiotic Factor A1 and Factor A2 has been determinedby using a Bruker Daltonics APEX II, 4.7 Tesla spectrometer fitted withan electrospray source. On the basis of these data, Factor A1 isassigned a molecular weight of 2246.71±0.06, calculated monoisotopicmass from [M+2H]²⁺ at m/z 1124.36124 (accuracy 30 ppm), determined byhigh resolution ESI-FTMS. Factor A2 is assigned a molecular weight of2230.71±0.06, calculated monoisotopic mass from [M+2H]²⁺ at m/z1116.36260 (accuracy 30 ppm), determined by high resolution ESI-FTMS.

Comparison of Antibiotic 107891 Factor A1 and Factor A2 with AntibioticsMF-BA-1768α₁ and MF-BA-1768β₁

A) Microbispora corallina NNRL 30420 (MF-BA-1768), described in U.S.Pat. No. 6,551,591 B1, was acquired from NNRL collection. In anexploratory experiment, the M. corallina NNRL 30420 (MF-BA-1768) strainhas been fermented in Erlenmeyer flask in the conditions described inU.S. Pat. No. 6,551,591 B1. The harvested broth was extracted bydilution with methanol. After centrifugation of the mycelium, thesupernatant was loaded on a HP20 polystyrenic absorption resin, elutedwith a methanol:water 70:30 mixture, which was reduced to small volumeand was then lyophilized.

In the chromatogram two peaks showed 1091 and 1108 [M+2H]²⁺ signals,corresponding to the [M+2H]²⁺ reported in U.S. Pat. No. 6,551,581 B1 forMF-BA-1768β₁ and MF-BA-1768α₁, respectively. The above extract was thenspiked with antibiotics 107891 Factors A1 and A2 and the mixture wasanalyzed by LC-MS. The peaks of antibiotics MF-BA-1768β1 andMF-BA-1768α1 and of antibiotics 107891 Factors A1 and A2 were found tohave distinct retention time and distinct [M+2H]²⁺ MS fragments.

B) In a further experiment, a 30 l thank fermentation of Microbisporasp. strain NRRL 30420 (MF-BA-1768) was performed and the harvested brothwas processed by following the description of U.S. Pat. No. 6,551,591B1. After purification steps sequentially on HP20 polystyrenic resin andpolyamide CC 6 0.1-0.3 mm (Macherey-Nagel) resin, two individualsubstances were obtained in pure form by preparative HPLC on a μ10particle size C18 Phenomenex (Torrance Calif., USA) Luna (250×12.2 mm)column eluted at flow rate 27 ml/min with the following multistepprogram: Time=0 min (32% of phase B); Time=8 min (32% of phase B);Time=20 min (36% of phase B); Time=32 min (90% Phase B). Phase A wasformic acid 0.05% (v/v) in water, Phase B was CH₃CN.

These substances showed antibacterial activity against staphylococci andenterococci as shown in Table IV. In LC-MS experiments the twosubstances showed [M+2H]⁺⁺ double protonated ions signals correspondingto antibiotic MF-BA-1768α1 and MF-BA-1768β1, as described in U.S. Pat.No. 6,551,591 B1.

TABLE IV MIC (μg/ml) MF-BA- MF-BA- 107891 107891 107891 STRAIN 1768α11768β1 A1 A2 complex 1400 0.13 0.5 0.13 0.13 0.13 Staphylococcus aureuscl. isol. Met r 568 4 16 1 2 2 Enterococcus faecium cl. isol. 569 4 8 12 2 Enterococcus faecium cl. isol. Van A 559 4 8 1 2 1 Enterococcusfaecalis cl. isol. 560 4 8 0.5 1 0.5 Enterococcus faecalis cl. isol. VanA

Experimental conditions of the antimicrobial tests were the same asthose utilized for the tests reported in Table VI below.

The LC-MS analyses of the isolated antibiotics MF-BA-1768α₁ andMF-BA-1768β₁ were performed on a Symmetry C₁₈ (5:m) 4.6×250 mm. column(Waters; Milford Mass., USA) equipped with a Symmetry C₁₈ (5:m) 3.9×20mm precolumn (both maintained in an oven at 50° C. temperature). Elutionwas performed at 1 ml/min flow rate with the following multistep elutionprogram: Time=0 min (30% Phase B); Time=8 min (30% Phase B); Time=20 min(45% Phase B); Time=24 min (90% Phase B); and Time=28 min (90% Phase B).Phase A was 25 mM HCOONH₄ buffer pH 4.5:CH₃CN 95:5 (v/v) and phase B wasCH₃CN. The HPLC equipment was coupled with a Finnigan LCQ ion trap MassSpectrometer (Thermoquest, Finnigan MAT, San Josè Calif., USA). 100μl/min of the effluents from the column were diverted to the ESIinterface of the LCQ Mass Spectrometer. The MS analysis was performedunder the following conditions: sample inlet: sheat gas flow (N₂) 25psi, aux gas flow 5 psi; capillary heater: 210° C.; sample inlet voltagepolarity both positive and negative; ion spray voltage: +/−4.75 KV;capillary voltage: +/−12 V; scan conditions: maximum ion time 50 ms;full micro: scan 3.

Individual antibiotic Factors MF-BA-1768α₁ and MF-BA-1768β₁ andantibiotics 107891 Factors A1 and A2 were analyzed individually and inmixture. The results are summarized in the following Table V.

TABLE V Ret. time (min) [M + 2H]²⁺ MF-BA-1768β₁ 12.86 1091 Antibiotic107891 A1 16.3 1124 Antibiotic 107891 A2 16.81 1116 MF-BA-1768α₁ 18.11108

In the same chromatographic system ramoplanin factor A2 (L. Gastaldo, R.Ciabatti, F. Assi, E. Restelli, J. K. Kettenring, L. F. Zerilli, G.Romanò, M. Denaro and B. Cavalleri, (1992): “Isolation, structuredetermination and biological activity of A-16686 Factors A′1, A′2 andA′3 glycolipodepsipeptide antibiotics”, J. Ind. Microbiol. 11: 13-18)was eluted with 11.00 min retention time.

NMR Spectroscopy of Antibiotic 107891 Factor A1 and Factor A2

¹H-NMR spectra of antibiotic 107891 Factor A1 and Factor A2 wererecorded in the mixture CD₃CN:D₂O (1:1) at 298 K on a Bruker AMX 600spectrometer applying a water suppression sequence. As internal standardthe residual signal of acetonitrile-d3 at 1.94 ppm was considered.

A) The ¹H-NMR spectrum of antibiotic 107891 Factor A1 is reported inFIG. 8.

¹H NMR spectrum of antibiotic 107891 Factor A1, dissolved in CD₃CN:D₂O(1:1), exhibits the following groups of signals (in ppm) at 600 MHzusing CD₃CN as internal standard (1.94 ppm), [δ=ppm, multiplicity;(attribution)]: 0.84 d (CH₃), 0.89 d (CH₃), 0.94 t (overlapped CH₃'s),1.1 d (CH₃), 1.13 d (CH₃), 1.15 t (overlapped CH₃'s), 149 m (CH₂), 1.69d (CH₃), 1.75 m (CH₂), 2.11 m (CH), 2.26 m (CH), 2.5 m (CH₂), 2.68-3.8 m(peptidic CH_(β)'s), 3.8-5.0 m (peptidic CH_(α)'s), 5.45-6.17 s (CH₂),5.58 d (CH double bond), 6.36 m (CH), 6.86 d (CH double bond), 7.0-7.45m (aromatic CH's). The dimethyl sulfoxide signal is present at 2.58 ppmand the formate signal is also present at 8.33 ppm as impurities.

B) The ¹H NMR spectrum bb decoupled of antibiotic 107891 Factor A2 isreported in FIG. 9.

¹H NMR spectrum of antibiotic 107891 Factor A2, dissolved in CD₃CN:D₂O(1:1), exhibits the following groups of signals (in ppm) at 600 MHzusing CD₃CN as internal standard (1.94 ppm), [δ=ppm, multiplicity;(attribution)]: 0.84 d (CH₃), 0.88 d (CH₃), 0.94 d (CH₃), 1.06 d (CH₃),1.14 d (CH₃), 148 m (CH₂), 1.65-1.75 m (CH₂), 1.67 d (CH₃), 2.15 m (CH),2.25 m (CH), 2.5 m (CH₂), 2.77-3.8 m (peptidic CH_(β)'s), 3.8-4.9 m(peptidic CH_(α)'s) 5.45-6.14 s (CH₂), 5.59 d (CH double bond), 6.34 m(CH), 6.84 d (CH double bond), 7.0-7.42 m (aromatic CH's). The dimethylsulfoxide signal is present at 2.58 ppm and the formate signal is alsopresent at 8.32 ppm as impurities.

¹³C-NMR spectra of antibiotic 107891 Factor A1 and Factor A2 wererecorded in the mixture CD₃CN:D₂O (1:1) at 298 K on a Bruker AMX 600spectrometer using as internal standard the residual signal ofacetonotrile-d3 at 1.39 ppm.

C) The ¹³C-NMR spectrum of antibiotic 107891 Factor A1 is shown in FIG.10. ¹³C NMR spectrum of antibiotic 107891 Factor A1, dissolved inCD₃CN:D₂O (1:1), exhibits the following groups of signals (in ppm) at600 MHz using CD₃CN as internal standard (1.39 ppm), [6=ppm;(attribution)]: 13.6-23.03 (aliphatic CH₃'s), 25.69-77.9 (aliphaticCH₂'s and peptidic CH_(α)'s), 105-137.3 (aromatic and double bonds CH'sand quaternary carbons), 165.6-176.6 (peptidic carbonyls).D) The ¹³C-NMR spectrum bb decoupled of antibiotic 107891 Factor A2 isshown in FIG. 11.

¹³C-NMR spectrum of antibiotic 107891 Factor A2, dissolved in CD₃CN:D₂O(1:1), exhibits the following groups of signals (in ppm) at 600 MHzusing CD₃CN as internal standard (1.39 ppm), [δ=ppm; (attribution)]:13.6-22.9 (aliphatic CH₃'s), 25.65-73 (aliphatic CH₂'s and peptidicCH_(α)'s), 105-137.3 (aromatic and double bonds CH's and quaternarycarbons), 165.7-176.1 (peptidic carbonyls).

UV and I.R. Spectra of Antibiotic 107891 Factor A1 and Factor A2.

A) The infrared spectrum of antibiotic 107891 Factor A1 recorded in KBrwith a Bruker FT-IR spectophotometer model IFS 48, exhibits absorptionmaxima at (cm⁻¹): 3294; 3059; 2926; 1661; 1529; 1433; 1407; 1287; 1114;1021. Infrared spectrum is reported in FIG. 12.B) The U.V. spectrum of antibiotic 107891 Factor A1 recorded inmethanol:H₂O 80:20 (v/v) with a Perkin-Elmer spectrophotometer Lambda16, exhibits two shoulders at 226 and 267 nm. U.V. spectrum is reportedin FIG. 13.C) The infrared spectrum of antibiotic 107891 Factor A2 recorded in KBrwith a Bruker FT-IR spectophotometer model IFS 48, exhibits absorptionmaxima at (cm⁻¹): 3296; 3060; 2928; 1661; 1529; 1433; 1407; 1288; 1116.Infrared spectrum is reported in FIG. 14.D) The U.V. spectrum of antibiotic 107891 Factor A2 recorded inmethanol:H₂O 80:20 (v/v) with a Perkin-Elmer spectrophotometer Lambda16, exhibits two shoulders at 226 and 267 nm. U.V. spectrum is reportedin FIG. 15.

On the basis of the physico chemical data reported above, the followingstructure formula can be assigned to antibiotic 107891:

wherein X is H or a halogen (F, Cl, Br, I), Y₁, Y₂, Y₃, Y₄, and Y₅ mayindependently be S, S—O³¹, S═O, O³¹ 13 S═O, and O═S═O, and wherein R₁,R₂, R₃, R₄, R₅, R₆, R₇, and R₈ may independently be H, OH, alkyl(branched or unbranched, substituted or unsubstituted), or aryl(substituted or unsubstituted).

In an alternative embodiment, R₁, R₂, R₃, and R₄ may be H or OH.Therefore, the possible combinations of R₁, R₂, R₃, and R₄ include thefollowing.

R₁ R₂ R₃ R₄ H H H H OH H H H H OH H H H H OH H H H H OH OH OH H H OH HOH H OH H H OH H OH OH H H H OH OH H OH H OH OH OH OH H OH OH H OH OH HOH OH H OH OH OH OH OH OH OHSimilarly, R₅, R₆, R₇, and R₈ may be H or OH. Therefore, the possiblecombinations of R₅, R₆, R₇, and R₈ include the following.

R₅ R₆ R₇ R₈ H H H H OH H H H H OH H H H H OH H H H H OH OH OH H H OH HOH H OH H H OH H OH OH H H H OH OH H OH H OH OH OH OH H OH OH H OH OH HOH OH H OH OH OH OH OH OH OH

Moreover, on the basis of the physico chemical data reported above, thefollowing structure formula can be assigned to antibiotic 107891 FactorA1, wherein X is Cl; Y₁, Y₂, Y₃, Y₄, and Y₅ are S; R₁ is H; R₂ is OH; R₃is H; and R₄, R₅, R₆, R₇, and R₈ are H, which is a preferred embodimentof the invention together with the pharmaceutically acceptable saltsthereof:

Additionally, on the basis of the physico chemical data reported above,the following structure formula can be assigned to antibiotic 107891Factor A2, wherein X is Cl; Y₁, Y₂, Y₃, Y₄, and Y₅ are S; R₁ is OH; R₂is OH; R₃ is H; and R₄, R₅, R₆, R₇, and R₈ are H, which is a preferredembodiment of the invention together with the pharmaceuticallyacceptable salts thereof:

In Vitro Biological Activity of Antibiotic 107891

Antimicrobial activity of the antibiotic 107891 was determined by thebroth microdilution method according to the National Committee forClinical Laboratory Standards recommendations (NCCLS, document M7-A5).

The strains used were clinical isolates or strains from American TypeCulture Collection (ATCC). The result of the tests are reported in TableVI and Table VII.

Antibiotic 107891 was dissolved in DMSO to obtain a 1000 μg/ml stocksolution, and subsequently diluted in water to obtain working solution.The media used were cation-adjusted Mueller Hinton broth (CAMHB) forStaphylococci, M. catarrhalis, Enterococci and L. monocytogenes; ToddHewitt broth (THB) for Streptococci; GC medium+1% Isovitalex+1% haeminefor Neisseria spp.; Brain Hearth Infusion+1% C supplement for H.influenzae; Lactobacillus broth for Lactobacilli; Middlebrook 7H9 withMiddlebrook OADC enrichment for M. smegmatis; RPMI 1640 Medium for C.albicans. Wilkins Chalgren broth+oxyrase (1:25 v/v) for Clostridia;Brucella broth containing cisteine (0.5 g/L) for Propionibacteria.Inocula for bacteria were 10⁵ CFU/ml. C. albicans inoculum was 1×10⁴CFU/ml. All the tests were performed in presence of 0.02% of bovineserum albumin (BSA). Cultures were incubated at 35° C. in air exceptClostridia and Propioniobacteria strains that needed anaerobicatmosphere. After 18-24 hours visual readings were performed and MICsdetermined. The MIC was defined as the lower concentration of antibioticat which there is no visible growth.

TABLE VI Antimicrobial activity of antibiotic 107891 MIC (μg/ml)Microorganism 107891 819 Staph. aureus Smith ATCC1936 ≦0.13 4061 Staph.aureus LIM1 ≦0.13 3798 Staph. aureus clin. isolate VISA 2 1400 Staph.aureus clin. isolate Met-R ≦0.13 613 Staph. aureus clin. isolate Met-R≦0.13 3797 Staph. aureus clin. isolate VISA Me 2 4064 Staph. aureus LIM2CHSA Met-R 0.5 1729 Staph. haemolyticus Met-R 8 1730 Met-S 2 147 Staph.epidermidis ATCC12228 ≦0.13 1139 4 44 Strept. pneumoniae Pen-S ≦0.132858 Pco-I ≦0.13 49 Strept. pyogenes ≦0.13 559 Ent. faecalis Van-S 1 560Ent. faecalis Van-A 0.5 A533 Ent. faecalis Van-A 1 568 Ent. faeciumVan-S 2 569 Ent. faecium Van-A 1 B518 Ent. faecium Van-A 2 A6345 Ent.faecium Van-A Lnz-R 4 3754 Mycobacterium smegmatis 32 E34 Listeriagarvias ≦0.13 148 Listeria delbrueckii ATCC4797 4 1450 Listeriamonocytogenes 0.125 833 Haemophilus influenzae 32 970 Hasmophilusinfluenzae ATCC 19418 32 3924 Moraxella catharralis 1 16 Moraxellacatharralis ATCC8176 0.25 1613 Neisseria meningitidis ATCC13090 0.5 997Neisseria gonorrhaee 0.25 47 Etcherichia coli >128 145 Candida albicans>128

TABLE VII Antimicrobial activity of antibiotic 107891 against anaerobesbacteria MIC (μg/ml) Microorganism Antibiotic 107891 ATCC 27520Propionibacterium limphophilum 0.015 ATCC 25564 Propionibacteriumgranulasum 0.03 ATCC 14157 Propionibacterium proptanicus 4 P9Propionibacterium acnes 0.125 1329 Propionibacterium acnes 0.5 ATCC25746 Propionibacterium acnes 0.015 ATCC 6919 Propionibacterium acnes0.125 ATCC 6922 Propionibacterium acnes ≦0.0039 ATCC 1348Propionibacterium acnes 0.25 4018 Clostridium difficile ≦0.125 4025Clostridium difficile ≦0.125 4022 Clostridium difficile ≦0.125 4032Clostridium perfringens ≦0.125 4043 Clostridium butyricum ≦0.125 4009Clostridium bestertuckii ≦0.125 4052 Clostridium septicum ≦0.125 60601Peptostreptococcus anaerobius >128

Antibiotic 107891 shows a good antibacterial activity againstGram-positive bacteria.

The MIC range against Staphylococcus spp., including MethicillinResistant (MRSA) and Glycopeptides Intermediate (GISA) resistantstrains, is =0.13-4 μg/ml and against recent clinical isolates ofEnterococcus spp., including Vancomycin Resistant (VRE), is 0.5-4 μg/ml.Against Streptococcus spp. MICs are ≦0.13 μg/ml.

Antibiotic 107891 is also active against anaerobic Gram-positivestrains; the MICs are ≦0.13 μg/ml against Clostridia and ≦0.004-4 μg/mlagainst Propionibacteria. Antimicrobial activities were showed againstL. monocytogenes (MIC 0.125 μg/ml) and Lactobacilli strains (MICs range≦0.13-4 μg/ml). Some Gram-negative bacteria are susceptible toantibiotic 107891; MICs are 1-0.25 μg/ml versus M. catharralis, 0.5-0.25μg/ml against Neisseria spp. and 32 μg/ml against H. influenzae.

Antibiotic 107891 is not active against the E. coli and C. albicansstrains tested.

In time-kill experiments antibiotic 107891 shows bactericidal activityagainst S. aureus GISA and E. faecalis VanA strain; at 24 hours thebactericidal concentration is the MIC value in Mueller Hinton broth.

S. aureus can cause life-threatening infections and MRSA is ofparticular clinical significance because it is resistant to allpenicillins and cephalosporins and also to multiple other antibiotics;in addition it easily spreads from patient to patient causing outbreaksof infection with important implications for healthcare facilities (W.Witte, (1999): “Antibiotic resistance in Gram-positive bacteria:epidemiological aspects”, Journal of Antimicrobial Chemotherapy 44:1-9).The Centers for Disease Control (CDC) National Nosocomial InfectionSurveillance System (NNIS) reported that methicillin resistance among S.aureus in US hospitals increased from 2.4% in 1975 to 29% in 1991, witha higher degree of resistance in intensive care units (L. Archibald, L.Philips, D. Monnet, J. E. Jr Mc Gowan, F. Tenover, R. Gaynes, (1997):“Antimicrobial resistance in isolates from inpatients and outpatients inthe United States: increasing importance of the intensive care unit”,Clinic Infect. Dis. 24: 211-5). Nosocomial staphylococcal infections areassociated with considerable morbidity and mortality, prolonging theduration of stay and increasing hospitalization costs. The majority ofMRSA strains are resistant to several of the most commonly usedantimicrobial agents, including macrolides, aminoglycosides, and theβ-lactams antibiotics in current use, including the latest generation ofcephalosporins.

Vancomycin resistant hospital-acquired pathogens responsible forinfections (such as endocarditis, meningitis and septicemia) are posingan increasing therapeutic challenge (Y. Cetinkaya, P. Falk and C. G.Mayhall, (2000): “Vancomycin-resistant enterococci”, Clin. Microbiol.Rev. 13: 686-707; L. B. Rice, (2001): “Emergence of vancomycin-resistantenterococci”,. Emerg. Infec. Dis. 7:183-7).

S. pneumoniae and M. catarrhalis are recognized important pathogens ofhumans. They are a common cause of respiratory tract infections,particularly otitis media in children and lower respiratory tractinfections in the eldery. M. catarrhalis and S. pneumoniae have beenrecently accepted as the commonest pathogens of the respiratory tract(M. C. Enright and H. McKenzy, (1997): “Moraxella (Branhamella)catarrhalis. Clinical and molecular aspect of a rediscovered pathogen”,J. Med. Microbiol. 46:360-71).

Clostridia are responsible of different diseases: gas gangrene andrelated wound infections, tetanus, botulism, antibiotic associateddiarrhea (CDAD) and pseumembranous colitis. Most of these microorganismsproduce exotoxins that play an important role in the pathogenesis of thediseases. C. difficile is the causative agent responsible for 25% ofcases of CDAD and for virtually all cases of pseudomembranous colitis.Over the last years the occurrence of C. difficile coinfection hasoccurred in patients with vancomycin resistant enteroccoccal infectionor colonization (J. G. Bartlett, (1992): “Antibiotic associateddiarrhea”, Clinic. Infect. Dis. 15: 573-581).

In Vitro Biological Activity of Antibiotic 107891 Factors A1 and A2

Table VIII reports the antimicrobial activities of the individualFactors A1 and A2 of antibiotic 107891. MICs were determined bymicrobroth dilution method as above described.

TABLE VIII Antimicrobial activity of antibiotic 107891 Factors A1 and A2MIC (μg/ml) Microorganism Factor A1 Factor A2 819 Staph. aureus Met-S≦0.03 ≦0.03 1524 Staph. aureus Met-R ≦0.03 ≦0.03 2235 Staph. aureusMet-R 0.06 0.06 3894 Staph. epidermidis Met-R ≦0.03 0.06 3881 Staph.epidermidis Met-R 0.06 ≦0.03 602 Staph. haemolyticus Met-R 0.25 0.253919 Strept. pneumoniae Pen-R ≦0.0015 ≦0.0015 3915 Strept. pneumoniaePen-S ≦0.0015 ≦0.0015 4323 Ent. faecalis VanA ≦0.03 ≦0.03 J1 Ent.faecalis VanA 1 1 4341 Ent. faecalis VanB 0.5 0.5 4397 Ent. faecalisVanB 1 1 4341 Ent. faecalis VanB 2 2 6349 Ent. faecium Van A LNZ-R 2 2 4Ent. faecium Van A 1 1 3 Ent. faecium Van A 0.5 0.5 D561 Ent. faeciumVan A 2 2 A8 Ent. faecium Van A 0.5 0.5 4339 Ent. faecium VanD 0.25 0.254174 Ent. gallinarum 1 1 997 Neisseria gonorrhaee 0.5 0.25 1613Neisseria meningitidis 0.25 0.25 1016 Propionibacterium. acnes <0.030.06

Biological Activity of Antibiotic 107891

Female ICR mice (Harlan Italia SpA—S. Pietro al Natisone, Italy)weighing 23-25 g were used in experiments of acute lethal infection inimmunocompetent or neutropenic mice. Neutropenia was induced by twointraperitoneal administrations of cyclophosphamide, 200 and 100 mg/kg,at four days and one day, respectively, before the mice were infected.

Infection was induced by inoculating intraperitoneally inimmunocompetent mice (8 animals/dose/treatment group) a bacterialsuspension of either a clinical isolate of methicillin resistantstaphylococcus (Staph. aureus SA3817) or a standard methicillinsusceptible strain (Staph. aureus Smith ATCC19636), or by inoculating inneutropenic mice a clinical isolate of glycopeptide resistantenterococcus (Ent. faecalis A533). The bacterial challenges (ca 10⁶cells/mouse) were given suspended in 0.5 mL of 5% bacteriological mucin(Difco). Untreated animals died within 24-72 h after infection.Antibiotic treatment began within 10-15 min after challenge. Antibiotic107891 was administered once intravenously or subcutaneously indifferent aqueous formulations. The 50% effective dose (ED₅₀) and 95%confidence limits were calculated by the Spearman-Kärber method (D. J.Finney, (1952): “The Spearman-Kärber method”, in: Statistical methods inbiological assay. pp. 524-530, Charles Griffin & Co., Ltd., London) fromthe percentage of animals surviving at day 7. Results are reported inthe following Table IX.

Antibiotic 107891 is not toxic up to the maximum tested dose of 200mg/kg.

TABLE IX ED₅₀s of antibiotic 107891 in acute lethal infections in mice.ED₅₀ 95% confidence Formulation Strain Route mg/kg limits A MSSA iv 2.11.7-2.7 sc 2.1 1.7-2.7 A VanA iv 3.2 2.7-3.9 sc 11.1  9.2-13.5 B MRSA sc4.2 3.5-5.1 C VanA iv 3.7 2.8-4.9 sc 12.7 10.7-15.0 Formulations: A: 10%(v/v) DMSO, 10% (w/v) Beta hydroxy-propyl cyclodextrin (Sigma), 80%(v/v) of 5% (w/v) glucose in H₂O B: 10% (v/v) DMSO, 40% (v/v) PEG 400 in0.1 M aqueous CH₃COOH C: 50% (v/v) PEG 400 in H₂O Strains: I. MSSA:Staph. aureus Smith 819 ATCC19636 II. MRSA: Staph. aureus 3817, clinicalisolate III. VanA: Ent. faecalis A533, clinical isolate, in neutropenicmice

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A (full scan low resolution spectrum) and 1B (zoom-scan highresolution spectrum) represent mass spectra of antibiotic 107891 showinga doubly protonated ion at m/z 1124 and m/z 1116.

FIG. 2 represents the I.R. absorption spectrum of antibiotic 107891dispersed in KBr.

FIG. 3 represents the UV spectrum of antibiotic 107891 dissolved inmethanol:H₂O.

FIG. 4 represents the ¹H-NMR spectrum recorded in the mixturemethanol-d4:H₂O (pH 4.3 HCl) 40:10 (v/v) at 40° C. on a Bruker AMX 600spectrometer applying a water suppression sequence.

FIG. 5 represents the ¹³C-NMR spectrum recorded in the mixturemethanol-d4:H₂O (pH 4.3 HCl) 40:10 (v/v) at 40° C. on a Bruker AMX 600spectrometer.

FIG. 6A (full scan low resolution spectrum) and 6B (zoom-scan highresolution spectrum) represent mass spectra of antibiotic 107891 FactorA1 showing a doubly protonated ions [M+2H]²⁺ at m/z 1124.

FIG. 7A (full scan low resolution spectrum) and 7B (zoom-scan highresolution spectrum) represent mass spectra of antibiotic 107891 FactorA2 showing a doubly protonated ions [M+2H]²⁺ at m/z 1116.

FIG. 8 represents the ¹H-NMR spectrum of antibiotic 107891 Factor A1recorded in the mixture CD₃CN:D₂O (1:1) at 298 K on a Bruker AMX 600spectrometer applying a water suppression sequence.

FIG. 9 represents the ¹H-NMR spectrum of antibiotic 107891 Factor A2recorded in the mixture CD₃CN:D₂O (1:1) at 298 K on a Bruker AMX 600spectrometer applying a water suppression sequence.

FIG. 10 represents the ¹³C-NMR spectrum of antibiotic 107891 Factor A1recorded in the mixture CD₃CN:D₂O (1:1) at 298 K on a Bruker AMX 600spectrometer.

FIG. 11 represents the ¹³C-NMR spectrum of antibiotic 107891 Factor A2recorded in the mixture CD₃CN:D₂O (1:1) at 298 K on a Bruker AMX 600spectrometer.

FIG. 12 represents the I.R. absorption spectrum of antibiotic 107891Factor A1 dispersed in KBr.

FIG. 13 represents the U.V. spectrum of antibiotic 107891 Factor A1dissolved methanol:H₂O.

FIG. 14 represents the I.R. absorption spectrum of antibiotic 107891Factor A2 dispersed in KBr.

FIG. 15 represents the U.V. spectrum of antibiotic 107891 Factor A2dissolved methanol:H₂O.

EXAMPLES Example 1 Fermentation Method of Microbispora sp. ATCC PTA-5024

Microbispora sp. ATCC PTA-5024 strain was maintained on oatmeal agarslants for 2-3 weeks at 28° C. The microbial content of one slant wasscraped with 5 ml sterile water and inoculated into 500 ml Erlenmeyerflasks containing 100 ml of seed medium (AF/MS) which is composed of(g/l): dextrose 20, yeast extract 2, soybean meal 8, NaCl 1 and calciumcarbonate 4. Medium was prepared in distilled water and pH adjusted to7.3 prior to sterilization at 121° C. for 20 min. The inoculated flaskswere grown at 28° C., on a rotatory shaker operating at 200 rpm. After4-6 days, 5% of this culture was inoculated into a second series offlasks containing the same fermentation medium. After 72 hours ofincubation, 200 ml were transferred into 4 l bioreactor containing 3 lof the same vegetative medium.

The fermentation was carried out at 30° C., with 700 rpm stirring and0.5 vvm aeration. After 72 hours the culture (1.5 l) was transferredinto a 20 l bioreactor containing 15 l of the same vegetative medium.The fermentation was carried out for 48 hours at 30° C., at 500 rpmstirring and at 0.5 vvm aeration and then was transferred to theproduction tank. The production of antibiotic 107891 was performed in a300 l fermenter containing 200 l of the production medium M8 composed of(g/l): starch 20, glucose 10, yeast extract 2, casein hydrolysed 4, meatextract 2 and calcium carbonate 3. The medium was prepared in deionizedwater and the pH adjusted to 7.2 before sterilization at 121° C. for 25min. After cooling the fermenter was inoculated with about 14 l (7%) ofpre-culture. Fermenter was run at 29° C., at 180 rpm stirring and at 0.5vvm aeration with a head pressure of 0.36 bar. The fermenter washarvested after 98 hours of fermentation.

The production of the antibiotic 107891 was monitored by HPLC aspreviously described, after extraction of the whole culture broth withthe same volume of methanol. The extraction was performed at roomtemperature under stirring for one hour.

Example 2 Alternative Fermentation Method of Microbispoza sp. ATCCPTA-5024

Microbispora sp. ATCC PTA-5024 was inoculated in 500 ml Erlenmeyerflasks containing 100 ml of growing medium (G1) consisting of g/l:glucose 10, maltose 10, soybean oil 10, soybean meal 8, yeast extract 2and calcium carbonate 4. The medium was prepared in deionised water andsterilized at 120° C.×20 min. without pH adjustment. The inoculatedflasks were incubated for 120-168 hours at 28° C., under 200 rpmstirring till a good growth was observed. The flasks were then used toinoculate (3%) a 4 l bioreactor containing 3 l of seed medium AF/MS,which is composed as described in Example 1. After 120 hours offermentation at 30° C., 700 rpm stirring and 0.5 vvm aeration, 1.5 l ofthe culture was transferred to a 20 l bioreactor containing 15 l of thesame vegetative medium. The fermentation was carried out for 96 hours at30° C., 600 rpm stirring and 0.5 vvm aeration, and was then transferredto the production tank.

The antibiotic production was obtained in a 300 l fermenter containing200 l of the productive medium (V6) consisting of (g/l): dextrose 20,yeast extract 5, meat extract 5, hydrolysed casein 3, peptone 5 and NaCl1.5. The medium was prepared in deionised water at pH adjusted to 7.5with NaOH, and was sterilized at 121° C. for 20 min.

The fermenter was inoculated with 14 l of seed culture (7%) and thefermentation was carried out at 29° C., stirred at 180 rpm, aerated with100 l of standard air per minute (0.5 vvm). The antibiotic 107891production was monitored by HPLC as previously described. Thefermentation was harvested after about 160 hours.

Example 3 Recovery of Antibiotic 107891

The fermentation broth described in the Example 1 was filtered bytangential filtration system (0.1 μm pore size membrane, Koch Carbo-Cor,Koch Wilmington, USA) to obtain 170 l of supernatant and 30 l ofconcentrated mycelium. Antibiotic 107891 complex was found both in thefiltrate (A) and in the mycelium (B).

(A) The filtered broth was stirred one night at room temperature in thepresence of Diaion HP-20 polystyrenic resin (4 l). The resin was thenrecovered, washed with 10 l methanol:water 4:6 (v/v) and elutedbatchwise initially with 10 l methanol:water 9:1 (v/v) and then with 10l methanol:butanol:water: 9:1:1 (v/v). The pooled eluted fractionscontaining antibiotic 107891 were concentrated to small volume on arotary evaporator and then were freeze-dried, yielding 32 g of rawmaterial. This raw material was dissolved in n-butanol (1 l) and thenextracted three times sequentially with 800 ml water. The organic layerwas concentrated under reduced pressure to an oily residue, which wasdissolved in methanol. Upon addition of petroleum ether, 5 g of crudeantibiotic preparation was obtained by precipitation.(B) After addition of 25 l of methanol, the retentate portion containingthe mycelium was stirred for 1 hour and was filtered to obtain 45 l ofmycelium extract. This solution was then diluted with water (20 l) andwas stirred one night at room temperature with Diaion HP-20 polystyrenicresin (1 l). The resin was then recovered, washed with 2 lmethanol:water 40:60 (v/v) and eluted batch-wise sequentially with 3 lmethanol:water 85:15 (v/v) and then with 2 l methanol:water 90:10 (v/v).The eluted fractions were monitored for the presence of antibiotic107891 by agar diffusion assay on Staphylococcus aureus and byanalytical HPLC method as previously reported.

The eluted fractions containing antibiotic 107891 were pooled, wereconcentrated under reduced pressure and were freeze dryed, yielding 8.1grams of crude antibiotic 107891.

Example 4 Alternative Recovery of Antibiotic 107891

The harvested broth from the 200 l tank fermentation described inexample 2 was brought to pH 6.8 and the broth was filtered by tangentialfiltration (0.1μ pore size membrane, Koch Carbo-Cor). The permeate (180l) was stirred batch-wise overnight at room temperature with 2 l ofDiaion HP20 resin (Mitsubishi Chemical) and the resin was thencollected.

Methanol (25 l) was added to the retentate portion in the tangentialfiltration equipment (about 20 l) containing the concentrated mycelium.This suspension was stirred for 1 hour and then was filtered with themicrofiltration system to a residual retentate volume of about 20 l.Additional methanol (25 l) was then added and the above process wasrepeated sequentially for a total of 5 cycles. The pooled methanolextracts (about 125 l) were diluted with 160 l of demineralized waterand were stirred batch-wise overnight at room temperature with 3 l ofDiaion HP 20 resin. The resin was then collected, and was pooled withthe Diaion HP 20 resin used to extract the broth permeate according tothe process above described. The pooled resin was washed into achromatographic column with 20 l of water:methanol 6:4 (v/v). Theantibiotic 107891 was eluted with 23 l of methanol: 50 mM ammoniumformate buffer pH 3.5: n-butanol 9:1:1 (v/v). This eluate was thenconcentrated under vacuum to a final volume of 3 l. The concentratedsolution was then loaded at pH 4.5 on a column of 2.5 l of polyamide CC6 0.1-0.3 mm (Macherey-Nagel) conditioned with water:methanol 7:3 (v/v).The column was washed with water:methanol 7:3 (v/v) and then with 25 mMammonium formate buffer pH 3.5: methanol 7:3 (v/v). The antibiotic waseluted with water:methanol 3:7 (v/v) and then with 1:9 (v/v) mixture.The elution was completed with 25 mM ammonium formate buffer pH 2.8:methanol in the ratio 1:9 (v/v). The eluates containing antibiotic107891 were pooled and concentrated under vacuum to a final volume of 1l. The pH of the concentrated solution was brought from 4 to 5.7 with 7M ammonium hydroxide and then the mixture was centrifuged to collect theprecipitate. This solid was suspended in water and freeze-dried,yielding 6.96 g of antibiotic 107891 preparation.

Example 5 Purification of Antibiotic 107891

Crude antibiotic 107891 (3.6 g), prepared as described in Example 3, waspurified by medium pressure chromatography on 100 g of reverse phase C8(EC) 40-70 μm particle size, 60A pore size, IST (International SorbentTechnology, Mid-Glamorgan, UK) by using a Bùchi B-680 Medium PressureChromatography System (Bùchi laboratoriums-technik AG, FlawilSwitzerland) equipped with B-687 gradient former, B-684 fractioncollector, B-685 glass column 70×460 mm. The resin was previouslyconditioned with a mixture of phase A: phase B 8:2 (v/v) and was theneluted at 25 ml/min with 60 min linear gradient from 20% to 60% of phaseB in 60 min.

Phase A was acetonitrile: 20 mM ammonium formate buffer (pH 6.6) 10:90(v/v); and phase B was acetonitrile: 20 mM ammonium formate buffer (pH:6.6) 90:10 (v/v).

The fractions containing antibiotic 107891 were pooled, concentratedunder vacuum and lyophilized twice from water, yielding 430 mg ofpurified antibiotic 107891.

Example 6 Purification of Antibiotic 107891 by Preparative HPLC

Antibiotic 107891 was further purified by preparative HPLC on a Hibarprepacked lichrosorb RP8 (7:m particle size) column RT 250-25 mm, Merck,by using a 25 minutes linear gradient elution from 30% to 45% of PhaseB, at 30 ml/min flow rate. Phase A was 25 mM ammonium formate buffer pH4.5:acetonitrile 95:5 (v/v) and Phase B was acetonitrile.

A sample of Antibiotic 107891 from example 5 (300 mg) was dissolved in1.5 ml 350:l of DMSO:formic acid 95:5 (v/v) and 300 μl were processedper chromatographic run. Antibiotic 107891 was typically eluted in 15-16minutes. The eluted fractions of 5 chromatographic runs, containingantibiotic 107891, were pooled and were concentrated under vacuum. Theresidual solution was lyophilised from water three times sequentially,yielding 31 mg of antibiotic 107891 as a white powder.

Example 7 Separation and Purification of Individual Factors A1 and A2 ofAntibiotic 107891

Factors A1 and A2 were separated and purified from the antibiotic 107891complex of Example 5 by preparative HPLC on a Symmetry Prep C18 (7 μmparticle size) column 7.8×300 mm Waters (Mildfold USA) using twodifferent elution programs.

A) Factor A1 was purified by a 25 minutes linear gradient elution from30% to 45% of Phase B, at 3.5 ml flow rate. Phase A was 25 mM ammoniumformate buffer pH 4.5:acetonitrile 95:5 (v/v) and Phase B wasacetonitrile. Purified antibiotic 107891 complex (15 mg) was dissolvedin 350 μl of DMSO:formic acid 95:5 (v/v) and was processed perchromatographic run. The A1 and A2 Factors were typically eluted in a11-13 minutes time frame. The eluted fractions were then analysed byHPLC under the analytical conditions described above. The fractions of14 chromatographic runs, containing pure antibiotic 107891 Factor A1,were pooled and were concentrated under vacuum. The residual solutionwas lyophilized from water three times sequentially, yielding 15 mg ofpure Factor A1 as a white powder.B) Factor A2 was purified by isocratic elution at 7 ml flow rate with100 mM ammonium formate buffer pH 4:acetonitrile 82.5:17.5 (v/v).Purified antibiotic 107891 complex (5 mg) was dissolved in 250 μl ofacetic acid:acetonitrile: 100 mM ammonium formate buffer pH 4 50:120:80(v/v) mixture and was processed per chromatographic run. The A1 and A2Factors were typically eluted in a 9-10 minutes time frame. The elutedfractions were then analysed by HPLC under the analytical conditionsdescribed above. The fractions of 20 chromatographic runs, containingpure antibiotic 107891 Factor A2, were pooled and were concentratedunder vacuum. The residual solution was lyophilized twice from wateryielding 8 mg of pure Factor A2 as a white powder.

1. A compound, isolated from Microbispora sp. ATCC PTA-5042, of the formula

wherein X is selected from the group consisting of H, F, Cl, Br, and I; wherein Y₁, Y₂, Y₃, Y₄, and Y₅ are independently selected from the group consisting of S, S—O, S═O, O—S═O, and O═S═O; and wherein R₁, R₂, R₃, R₄, R₅, R₆, R₇, and R₈ are independently selected from the group consisting of H, OH, alkyl, and aryl.
 2. The compound of claim 1, wherein R₄, R₅, R₆, R₇, and R₈ are H.
 3. The compound of claim 2, wherein R₁ is H, R₂ is H, and R₃ is H.
 4. The compound of claim 2, wherein R₁ is H, R₂ is H, and R₃ is OH.
 5. The compound of claim 2, wherein R₁ is H, R₂ is OH, and R₃ is OH.
 6. The compound of claim 2, wherein R₁ is OH, R₂ is OH, and R₃ is OH.
 7. The compound of claim 2, wherein R₁ is OH, R₂ is H, and R₃ is H.
 8. The compound of claim 2, wherein R₁ is OH, R₂ is H, and R₃ is OH.
 9. The compound of claim 1, wherein Y₁ is selected from the group consisting of S—O, S═O, O—S═O, and O═S═O, and Y₂, Y₃, Y₄, and Y₅ are S.
 10. The compound of claim 1, wherein Y₂ is selected from the group consisting of S—O, S═O, O—S═O, and O═S═O, and Y₁, Y₃, Y₄, and Y₅ are S.
 11. The compound of claim 1, wherein Y₃ is selected from the group consisting of S—O.sup.-, S═O, O—S═O, and O═S═O, and Y₁, Y₂, Y₄, and Y₅ are S.
 12. The compound of claim 1, wherein Y₄ is selected from the group consisting of S—O.sup.-, S═O, O—S═O, and O═S═O, and Y₁, Y₂, Y₃, and Y₅ are S.
 13. The compound of claim 1, wherein Y₅ is selected from the group consisting of S—O.sup.-, S═O, O—S═O, and O═S═O, and Y₁, Y₂, Y₃, and Y₄ are S. 